Summary Pulmonary arterial hypertension (PAH) is a rare chronic human disease exhibiting high morbidity and mortality rates. Current treatments offer very limited benefits in terms of quality of life improvement and longevity to PAH patients. The expression of the gene TMEM16A or Anoctamin1 (ANO1) that encodes for Ca2+-activated Cl- channels (CaCCs) is enhanced in pulmonary artery smooth muscle cells (PASMCs) from animal models of pulmonary hypertension (PH). ANO1-encoded CaCCs are believed to act as an excitatory mechanism in response to vasoconstrictors such as serotonin. Based on new preliminary data gathered from mice expressing a Ca2+ biosensor protein called GCaMP3 only in smooth muscle cells, the postulated mechanism of excitation-contraction coupling (EC-coupling) linking the activity of vasoconstrictors to ANO1 needs to be revisited on the basis that the very stable contraction of pulmonary arteries produced by serotonin relies on a fine balance between highly localized cyclical SR Ca2+ release and reuptake, ANO1-mediated membrane depolarization and Ca2+ entry through CaV1.2 channels. How EC-coupling is altered and the role of increased ANO1 expression and function in PH is unknown. We recently reported that the activity of CaCCs in pulmonary artery smooth muscle cells is inhibited by direct binding of phosphatidylinositol 4,5 bisphosphate (PIP2), a key membrane phospholipid regulating many membrane proteins including ion channels and transporters. This discovery is significant, albeit controversial, because in the context of this new paradigm activation of ANO1 would be physiologically triggered by a dual self-reinforcing mechanism following stimulation of receptors leading to vasoconstriction in the pulmonary circulation. Preliminary data in this application show that ANO1 and several key enzymes regulating PIP2 levels are elevated in the chronic hypoxic mouse model of PH. The structural arrangement of the enzymes controlling PIP2 levels and ANO1 regulation, the impact of this relationship on pulmonary arterial tone and the domain(s) of ANO1 that interact with PIP2 are unknown. Using a multidisciplinary approach and several sophisticated transgenic conditional ANO1 knockout animal models and mouse and human PASMCs, we will test the hypothesis that the expression, function and regulation by PIP2 of ANO1 channels are altered and play a key role in the functional remodeling of PASMCs in chronic hypoxia-induced PH. Three specific aims are proposed to test this hypothesis: Specific Aim 1: To determine the role of ANO1 channels in the vasoconstriction and localized Ca2+ oscillations elicited by agonists in the PA from normal and PH mice. Specific Aim 2: What is the structural organization and functional significance of the ANO1 channel microdomain and regulation by PIP2 metabolism in PH? Specific Aim 3: What are the biophysical and molecular mechanisms involved in the modulation by PIP2 of native ANO1 channels in PASMCs and HEK-293 cells over-expressing ANO1?